This project aims to understand how bacterial proteins penetrate cell membranes and enter the cytoplasm of mammalian cells in order to understand how proteins can cross membranes. The primary subject of study is diphtheria toxin. Its membrane insertion occurs after exposure to the low pH within the lumen of endosomal vacuoles. The A chain of the toxin then translocates into the cytoplasm, aided by the toxin's hydrophobic T domain. To analyze this process, the structure of membrane-inserted A and T subunits will be studied, making use of site-directed mutagenesis combined with in vitro fluorescence-based assays. In addition, function will be evaluated by pore formation, translocation assays and cellular toxicity measurements. The role of the T chain will be analyzed by assessing the effects of blocking the insertion of individual T domain helices on membrane-inserted structure and function. Also, crucial T domain residues will be identified and their effect on structure and function characterized. The translocation mechanism will be studied by examining the role of pore formation, oligomerization, and the covalent link between the A and T chain on translocation. In addition, the role of interactions between the A and T chain in translocation will be analyzed by comparing their topography in the membrane-inserted A-T complex before, after and during translocation. Finally, the hypothesis that the T domain promotes translocation by acting like a relatively non-specific transmembrane chaperone will be tested by assaying the translocation of chimeras with the A chain replaced by proteins that vary in their degree of folding and/or hydrophobicity. Studies will then be extended to the type III translocation system of pathogenic bacteria. Interesting parallels between diphtheria toxin and type III translocation have recently become apparent. Our first target will be the YopB and YopD proteins of Yersinia. They are main components of the membrane-perforating apparatus through which translocating proteins pass. Our first goal will be to understand the relationship of YopB/D function to their topography when in the membrane-inserted state, with a longer-range goal being exploration of how YopB/D interactions with other Yersinia proteins results in the assembly of a functional translocation pore. An ultimate goal is to aid development of therapeutic agents interfering with infection. In a first step, the effects of molecules found to inhibit T domain pore formation upon YobB/D and Yersinia pathogenesis will be tested.